Project Summary Microrchidia 3 (MORC3) has been linked to cancer-associated inflammations. Recent studies from our group demonstrate that MORC3 is deregulated in patients with Down syndrome (DS), however the precise role of MORC3 in disease or in normal cellular processes remains poorly understood. Our preliminary data show that MORC3 has an intrinsic ATPase activity and that the catalytic ATPase domain binds to DNA, whereas the adjacent CW domain recognizes histone H3 and mediates the ATPase activity of MORC3. The molecular mechanisms underlying these novel functions of MORC3 are unclear and will be elucidated in the proposed studies. We hypothesize that the DNA-stimulated ATPase activity of MORC3 is mediated through histone-binding activity of the CW domain, which in the MORC3 inactive state, binds to and autoinhibits the ATPase domain; and that PTMs of histone H3 alter binding of CW and fine-tune the ATPase activity of MORC3. We aim to define the molecular basis and functional significance of the multivalent engagement of MORC3 with chromatin and to understand the mechanism of its autoregulation. By establishing the biological role and obtaining the mechanistic details, we will learn how this fundamental chromatin remodeling component can be controlled. In this research we will employ a powerful combination of in vitro and in vivo approaches to examine a newly identified epigenetic regulatory mechanism. We integrate X-ray crystallographic, advanced Frster Resonance Energy Transfer, NMR spectroscopic and high- throughput technological experiments with molecular and cell biology tools to gain insight into the role of the ATPase and CW domains in biological activities of MORC3. In-depth structural, biochemical and functional characterization of MORC3 will have significant impact on chromatin biology and basic molecular biology of chromatin-remodeling enzymes. It will also aid to our understanding of the epigenetic-driven signaling events that are deregulated in DS and other human diseases.